Forming sample combinations using liquid bridge systems

ABSTRACT

The present invention generally relates to methods of constructing liquid bridges and methods of forming predetermined combinations of samples using liquid bridges.

RELATED APPLICATIONS

The present invention is a continuation-in-part of U.S. nonprovisionalpatent application Ser. No. 12/092,261, filed Apr. 30, 2008, which is aU.S. national phase patent application from PCT international patentapplication number PCT/IE2007/000013, filed Feb. 7, 2007, which claimspriority to U.S. provisional patent application Ser. No. 60/765,671,filed Feb. 6, 2007, each of which is incorporated by reference herein inits entirety.

FIELD OF INVENTION

The present invention generally relates to methods of constructingliquid bridge systems and methods of forming predetermined combinationsof samples using liquid bridge systems.

BACKGROUND

Microfluidics involves micro-scale devices that handle small volumes offluids. Because microfluidics can accurately and reproducibly controland dispense small fluid volumes, in particular volumes less than 1 μl,application of microfluidics provides significant cost-savings. The useof microfluidics technology reduces cycle times, shortenstime-to-results, and increases throughput. Furthermore, incorporation ofmicrofluidics technology enhances system integration and automation.

Given the small dimensions of microfluidic devices or componentsthereof, these devices involve construction and design that differs frommacro-scale devices. Simple scaling down in size of conventional scaledevices to a microfluidic scale is not a design option. For example,liquid flow in microfluidic devices differs from that of macro-scalesize devices. Because liquid flow tends to be laminar, surface flux andsurface tension start to dominate and as a result, effects not seen atthe macro level become significant at the microfluidic level. Otherdifferences at the microfluidic level include faster thermal diffusion,predominately laminar flow, and surface forces that are responsible forcapillary phenomena.

There is an unmet need for improved microfluidic devices and systems andmethods of generating microfluidic samples.

SUMMARY

The invention generally relates to methods of using liquid bridges inorder to facilitate mixing multiple samples. A liquid bridge is a devicein which liquid droplets containing a sample of interest are formed. Thedroplets formed in a liquid bridge are enveloped in an immisciblecarrier fluid. A typical liquid bridge of the invention is formed by aninlet in communication with a chamber that is filled with a carrierfluid. The carrier fluid is immiscible with sample droplets flowingthrough the inlet into the chamber. The sample droplet expands until itis large enough to span a gap between the inlet and an outlet incommunication with the chamber. Droplet formation is accomplished inmany ways, for example, by adjusting flow rate or by joining a secondsample droplet to the first sample droplet, resulting in formation of anunstable funicular bridge that subsequently ruptures from the inlet.After rupturing from the inlet, the sample droplet enters the outlet,surrounded by the carrier fluid from the chamber.

The invention provides methods of using liquid bridges in order tocreate a sample array that allows mixing of a predetermined number ofdifferent samples. The invention provides methods for constructing aliquid bridge system having a predetermined number of liquid bridgessufficient for matrix combinations of any number of samples. In apreferred embodiment, a first sample array is combined with a secondsample array. Aspects of the invention are accomplished by ascertaininga number of wells within the first array of samples, and ascertaining anumber of wells within the second array of samples. For example, thefirst array and the second array may each independently be a 96 wellplate or a 384 well plate. The first array and the second array may eachindependently be an array having any number of wells (e.g., from about 2to about 5000).

Based on the number of wells in each of the first and second samplearray, a formula a_(n)×b_(n) is applied, in which a_(n) is the number ofwells in the first array and b_(n) is the number of wells in the secondarray. The output of this formula determines the number of liquidbridges needed to combine a first sample array and a second sample arrayto obtain the desired number of sample combinations. For example, if afirst array has four wells and a second array has four wells, then asystem would be constructed having 16 liquid bridges. Alternatively if afirst array has twelve wells and a second array has twelve wells, then asystem would be constructed having 144 liquid bridges. Not all of thewells of each array are required to be filled with a sample. Forexample, at least one of the sample combinations can be a combination ofa blank from the first array and a blank from the second array. Anexemplary blank is oil, e.g., silicone oil.

The first and second array of samples can each independently be chemicalor biological species. For example, the first array of samples can beprimers for PCR reactions and the second array of samples can includenucleic acid (e.g., DNA or cDNA) from a biological sample to beamplified by PCR.

In another aspect, the invention provides a method for formingpredetermined sample combinations including, providing a first samplearray, providing a second sample array, and providing at least oneliquid bridge to mix the first sample array with the second samplearray, wherein the number of liquid bridges provided is determined by aformula a_(n)×b_(n), wherein a_(n) is the number of wells in the firstarray and b_(n) is the number of wells in the second array.

The invention also provides a system for mixing samples, the systemincluding, a first gas-free sampling device that interacts with a firstsample array, a second gas-free sampling device that interacts with asecond sample array, and at least one liquid bridge for mixing the firstsample array with the second sample array, wherein the number of liquidbridges provided is determined by a formula a_(n)×b_(n), wherein a_(n)is the number of wells in the first array and b_(n) is the number ofwells in the second array.

The system can further include robotics to move the first and secondsampling devices to interact with the first and second arrays ofsamples, pumps for acquiring samples in the first and second arrays, acomputer operably connected to the system, and a thermocycler.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a drawing depicting an exemplary embodiment of a liquid bridgehaving two inlets and one outlet.

FIG. 2 is a drawing depicting another exemplary embodiment of a liquidbridge having two inlets and one outlet.

FIG. 3 is a drawing depicting an exemplary embodiment of a liquid bridgehaving one inlet and two outlets.

FIG. 4 is a schematic diagram depicting an exemplary liquid bridgesystem constructed according to the methods of the invention.

FIG. 5 is a schematic diagram depicting another exemplary liquid bridgesystem constructed according to the methods of the invention.

FIG. 6 is a schematic diagram depicting a matrix for forming samplecombinations.

FIG. 7 is a sequence of photographs showing liquid dynamics anddimensions at a liquid bridge.

FIG. 8 is a diagram showing a characteristic plot of volumetric ratiovs. slenderness at a liquid bridge for segmentation.

FIG. 9 is a set of photographs of liquid bridge segmentors havingdifferent geometries.

FIG. 10 is a characteristic plot for a liquid bridge segmentor.

FIG. 11 is a collapsed data characteristic plot for liquid bridgesegmentation.

FIG. 12 is a further characteristic plot for a liquid bridge segmentor.

FIG. 13 is a set of photographs of three liquid bridge segmentors havingdifferent capillary radii.

FIG. 14 is a further characteristic plot for a liquid bridge segmentor.

FIG. 15 is a photograph of a funicular bridge, also showing dimensionparameters.

FIG. 16 is a funicular liquid bridge characteristic stability plot.

DETAILED DESCRIPTION

An aspect of the invention provides methods for constructing a liquidbridge system having a predetermined number of liquid bridges sufficientto combine multiple sample arrays for analysis. In its simplest form,aspects of the invention are accomplished by ascertaining a number ofwells within a first array of samples, and ascertaining a number ofwells within a second array of samples. Based on the number of wells ineach of the first and second sample array, a formula a_(n)×b_(n) isapplied, in which a_(n) is the number of wells in the first array andb_(n) is the number of wells in the second array. The output of thisformula determines the number of liquid bridges needed to combine afirst sample array and a second sample array to obtain the desirednumber of sample combinations.

As used herein, an array refers to any device capable of holding asample. An array can be a plate, such as a 96 well microtiter plate or a384 well microtiter plate. An array can also be a single vessel or a setof vessels. The vessel can be any type of vessel that is suitable forholding a sample. Exemplary vessels include eppendorf tubes, vials,beakers, flasks, centrifuge tubes, capillary tubes, cryogenic vials,bags, channels, cups, or containers. A well refers to a portion of thearray that holds a sample, such as a well of a microtiter plate, a wellof an eppendorf tube, a well of a beaker, a well of a centrifuge tube,or a well of a bag.

The first and second array of samples can each independently be anychemical or biological species. In certain embodiments, the sample is agene or gene product from a biological organism. Standard scientificprotocols are available for extraction and purification of mRNA andsubsequent production of cDNA. In other embodiments, the sample includesPCR reagents. A typical Q-PCR reaction contains: fluorescentdouble-stranded binding dye, Taq polymerase, deoxynucleotides of type A,C, G, and T, magnesium chloride, forward and reverse primers and subjectcDNA, all suspended within an aqueous buffer. Reactants, however, may beassigned into two broad groups: universal and reaction specific.Universal reactants are those common to every Q-PCR reaction, andinclude: fluorescent double-stranded binding dye, Taq polymerase,deoxynucleotides A, C, G and T, and magnesium chloride. Reactionspecific reactants include the forward and reverse primers and patientcDNA.

FIG. 4 shows a schematic diagram of an exemplary liquid bridge systemconstructed according to the methods of the invention. First samplearray 25 contains four sample wells A1-A4 and second sample array 26contains four sample wells B1-B4. Based on the number of wells in thefirst sample array 25 and the number of wells in the second sample array26, a liquid bridge system is constructed having a number of liquidbridges sufficient to combine the first sample array and the secondsample array for analysis. In the embodiment shown in FIG. 4, a systemis constructed having 16 liquid bridges, thus providing a system withthe capacity to form the required number of sample combinations from afirst sample array having four wells and a second sample array havingfour wells. Samples of the first and second arrays are acquired withsampling devices, such as those shown in Davies et al. (U.S.nonprovisional patent application Ser. No. 12/468,367, filed May 19,2009, and titled “Sampling Devices”, the contents of which areincorporated by reference herein in their entirety).

For simplicity, FIG. 4 is limited to showing combinations of sample A1from the first sample array 25 with samples B1-B4 of the second samplearray 26. Sample A1 is mixed with sample B1 at liquid bridge 27, sampleA1 is mixed with sample B2 at liquid bridge 28, sample A1 is mixed withsample B3 at liquid bridge 29, and sample A1 is mixed with sample B4 atliquid bridge 30. Thus in any pair of positions of the first and secondsample arrays, all combinations of samples from the first array aremixed with samples from the second array.

FIG. 5 is a schematic diagram depicting another exemplary liquid bridgesystem constructed according to the methods of the invention. Firstarray 31 contains one sample well A1 and second array 32 contains foursample wells B1-B4. Based on the number of wells in the first samplearray 31 and the number of wells in the second sample array 32, a liquidbridge system is constructed with 4 liquid bridges. A system having 4liquid bridges has the capacity to form the required number of samplecombinations from a first sample array having one well and a secondsample array having four wells. Samples of the first and second arraysare acquired with sampling devices, such as those shown in Davies et al.(U.S. nonprovisional patent application Ser. No. 12/468,367, filed May19, 2009, and titled “Sampling Devices”).

FIG. 5 shows combinations of sample A1 from the first sample array 31with samples B1 through B4 of the second sample array 32. Sample A1 ismixed with sample B1 at liquid bridge 33, sample A1 is mixed with sampleB2 at liquid bridge 34, sample A1 is mixed with sample B3 at liquidbridge 35, and sample A1 is mixed with sample B4 at liquid bridge 36.Thus in any pair of positions of the first and second sample arrays, allcombinations of samples from the first array are mixed with samples fromthe second array.

Sampling devices can be traversed over arrays of any size, for example,96 well plates or 384 well plates, to give the desired combinations ofsamples from the first array with samples from the second array. Methodsof the invention can be used to construct a liquid bridge system of anysize. Additional exemplary constructed liquid bridge systems include: afirst array having two wells, a second array having two wells, and asystem having four liquid bridges; a first array having six wells, asecond array having six wells, and a system having 36 liquid bridges; afirst array having eight wells, a second array having eight wells, and asystem having 64 liquid bridges; a first array having 10 wells, a secondarray having 10 wells, and a system having 100 liquid bridges; or afirst array having 12 wells, a second array having 12 wells, and asystem having 144 liquid bridges, etc.

A matrix can be used to describe the combinations formed in a generalcase of mixing a first sample array with a second sample array, as shownin FIG. 6. Contents of wells of the first sample array are representedby (a), and contents of the wells of the second sample array arerepresented by (b). Mixing combinations are described as follows:

$\begin{matrix}{{a_{1} + b_{1}},{a_{2} + {b_{2,}\mspace{14mu} \ldots \mspace{14mu} a_{1}} + b_{n}}} \\{{a_{2} + b_{1}},{a_{2} + {b_{2,}\mspace{14mu} \ldots \mspace{14mu} a_{2}} + b_{n}}} \\\vdots \\\vdots \\\vdots \\\vdots \\\vdots \\{{a_{n} + b_{1}},{a_{2} + {b_{2,}\mspace{14mu} \ldots \mspace{14mu} a_{n}} + b_{n}}}\end{matrix}$

Thus the combinations required by the instrument user may therefore bespecified as a matrix. Further, not all of the wells of each array arerequired to be filled with a sample. For example, at least one of thesample combinations can be a combination of a blank from the first arrayand a blank from the second array. An exemplary blank is oil, e.g.,silicone oil. By utilizing blanks, the number of assays need not beequal to the number of samples. Further, the system preserves samples bymaking combinations of blanks (0+0), as opposed to making combinationsof a sample and a blank (a+0) or (b+0).

Mixing of samples from the first array with samples from the secondarray is accomplished using liquid bridges. Exemplary liquid bridges areshown in Davies et al. (WO 2007/091228, the contents of which areincorporated by reference herein in their entirety). In certainembodiments, a liquid bridge includes a chamber having at least oneinlet and at least one outlet. The chamber can include as many inletsand outlets as are desired, for example, one inlet and one outlet, twoinlets and two outlets, three inlets and three outlets, four inlets andfour outlets, one inlet and two outlets, one inlet and three outlets,one inlet and four outlets, two inlets and one outlet, etc.

The chamber and the inlets and outlets can be composed of any inertmaterial that does not interact with the sample or the carrier fluid.Exemplary materials include polytetrafluoroethylene (PTFE; commerciallyavailable from Dupont, Wilmington, Del.), polyetheretherketone (PEEK;commercially available from TexLoc, Fort Worth, Tex.), perfluoroalkoxy(PFA; commercially available from TexLoc, Fort Worth, Tex.), orFluorinated ethylene propylene (FEP; commercially available from TexLoc,Fort Worth, Tex.).

The chamber is configured to receive a carrier fluid, the carrier fluidfilling a space in the chamber between the inlet and the outlet. Thecarrier fluid is immiscible with the sample. In embodiments in which thesample is hydrophilic, an exemplary carrier fluid is an oil, for examplesilicone oil. In certain embodiments, the silicone oil is PD5 oil. Inother embodiments, the oil is any oil that contains a phenol group.Alternatively, the sample can be hydrophobic and exemplary carrierfluids include water or alcohol such as methanol or ethanol.

In certain embodiments, the carrier fluid is density matched with thesample such that a neutrally buoyant environment is produced within thechamber. In embodiments in which the carrier fluid is an oil, the oiltypically provides a pressure of no more than 0.5 to 1.0 bar aboveatmospheric pressure. The oil generally has a viscosity of about 0.08Pas to about 0.1 Pas.

The inlets and outlets can be of any shape, for example, circular,rectangular, triangular, or square. The inlets and outlets can have aninner diameter ranging from about 10 μm to about 3 mm. For example, theinlets an outlets have an inner diameter of about 10 Mm, about 50 μm,about 100 μm, about 150 μm, about 200 μm, about 400 μm, about 600 μm,about 900 μm, about 1 mm, about 2 mm, or about 3 mm. In certainembodiments, the inlets and outlets have the same inner diameter. Inother embodiments, the inlets and outlets have different innerdiameters. In certain embodiments, each of the inlets have differentinner diameters. In certain embodiments, each of the outlets havedifferent inner diameters.

The inlet(s) and outlet(s) have dimensions and are positioned in thechamber such that a sample periodically bridges from the inlet(s) to theoutlet(s), and droplets of the sample are periodically delivered to theoutlet(s). FIG. 1 shows an exemplary embodiment of a liquid bridgehaving two inlets and one outlet. Referring to FIG. 1 panel A, a bridge1 includes a first inlet 2, a narrower second inlet 3, an outlet 4, anda chamber 5. The chamber is filled with a carrier fluid, e.g., siliconeoil, and the carrier fluid is density-matched with the first sample 6such that a neutrally buoyant environment is created within the chamber5. The oil within the chamber is continuously replenished by the oilseparating formed droplets of sample. Replenishment of the oilseparating the formed droplets results in the droplets assuming a stablecapillary-suspended spherical form upon entering the chamber 5.

FIG. 1 panels B and C show that the spherical shape of the sample growsuntil large enough to span the gap between the ports, forming anaxisymmetric liquid bridge. FIG. 1 panel D shows that introduction of asecond sample droplet 7 from the second inlet 3 results in formation ofan unstable funicular bridge. FIG. 1 panel E shows that the unstablefunicular bridge quickly ruptures from the second inlet 3, and the firstand second sample droplets combine at the liquid bridge 1. FIG. 1 panelsF and G show that upon combination with the first sample 6 and thesecond sample 7, the droplet 8 containing each of the first sample 6 andthe second sample 7 ruptures from the first inlet 2 and enters theoutlet 4.

In further detail, the first inlet 2 and the outlet 3 are of diameter200 μm. The separation of the inlet 2 and the outlet 4 is about 1 mm.The second inlet 3 is of diameter 100 μm, and the distance between thesecond inlet 3 and the axis of the inlet 2 and the outlet 4 is 1.5 mm.The chamber 5 is 5 mm in diameter and 3 mm in depth. The carrier fluid,e.g., oil provides a pressure of no more than 0.5 to 1.0 bar aboveatmospheric, and has a viscosity of 0.08 to 0.1 Pas. The flow rate ofthe samples 6 and 7 entering chamber 5 is in the range of 2 μl/min to 5μl/min. The carrier fluid is density-matched with each of samples 6 and7 such that a neutrally buoyant environment is created within thechamber 5.

The pressure in the chamber 5 is atmospheric. The interfacial tensionwithin the chamber 5 is important for effective mixing of samples 6 and7. Also, the relative viscosity between the samples and carrier fluid isimportant. The internal pressure (Laplace pressure) within each dropletis inversely proportional to the droplet radius. Thus there is a higherinternal pressure within the droplet at the second inlet 3. Becausesample 6 and sample 7 are of the same phase, there is little interfacialtension between the droplets of these fluids. Thus, the internalpressures cause a joining of the droplets, akin to injection of one intothe other. Also, physical control of the locations of the sampledroplets 6 and 7 is achieved by the carrier fluid, which is immisciblewith the droplets. In certain embodiments, a surfactant can be added toeither the samples 6 and 7 or the carrier fluid to change theinterfacial tension.

FIG. 2 shows another exemplary embodiment of a liquid bridge having twoinlets and one outlet. Referring to FIG. 2 panel A, liquid bridge 9includes a first inlet 10, a second inlet 11, an outlet 12, and achamber 13. The chamber 13 is filled with a carrier fluid, e.g.,silicone oil. The chamber 13 is 5 mm in diameter and 3 mm in depth, andthe internal pressure caused by flow of carrier fluid, e.g., siliconeoil, from the second inlet 11 into the chamber 13 is no more than 0.5bar to 1.0 bar above atmospheric pressure. The diameter of the inlets 10and 11 and outlet 12 is 200 μm. The spacing between the first inlet 10and the outlet 12 is 0.5 mm. The spacing between these ports can rangefrom 0.2 mm to 1.5 mm. The flow rate of the sample from the inlet 10into the chamber 13 is 5 μl/min. The flow rate can generally range fromabout 2 μl/min to about 8 μl/min.

The geometry between liquid bridge 9, and the carrier fluid create aperiodic instability between the inlet 10 and the outlet 12 due tosurface tension. FIG. 2 panel A shows that an sample droplet 14 isinitially formed at the end of the inlet 10. As shown in FIG. 2 panel B,the sample droplet 14 momentarily bridges between the inlet 10 and theoutlet 12. The volume held in this bridge is then steadily reduced bythe action of pumping carrier fluid into the chamber through the secondinlet port 11. FIG. 2, panels B and C show that pumping carrier fluidinto the chamber while the sample droplet 14 momentarily bridges betweenthe inlet port 10 and the outlet port 12 results in the formation of anunstable liquid bridge that ruptures to release a microfluidic plug 15of sample that enters the outlet 12. FIG. 2 panel D shows thatsubsequent to rupture of the microfluidic plug 15, the process repeatsitself with the formation of another sample droplet 16 at the end ofinlet 10.

When the flow rate of the carrier fluid entering the chamber 13 frominlet port 12 is substantially the same as the flow rate of sampleentering the chamber 13 from the inlet port 10, smaller segmenteddroplets, separated by the same volume of carrier fluid, e.g., siliconeoil, are produced by the bridge 9. The segmenting mechanism reliablyproduces uniform aqueous microfluidic plugs separated by carrier fluidthat do not rely on the shear force exerted by the carrier fluid.

In another embodiment, mixing of sample droplets may be achieved using aconfiguration in which a chamber includes one inlet and two outlets.Sample droplets entering the chamber through the inlet are closetogether, and the delay for droplet formation within the chamber due toa reduction in fluid flow through a main line results in a collision andhence mixing. Such mixing may be caused by withdrawal of oil from thechamber, or upstream of it. Referring to FIG. 3, a liquid bridge 17 hasan inlet 18, a first outlet 19, a second outlet 20, and a chamber 21.The chamber is filled with carrier fluid, e.g., oil, that is immisciblewith the sample. A leading droplet of sample entering the chamber 21through the inlet 18 forms a sample droplet 22 in the chamber at the endof the inlet 18. FIG. 3 panels B and C show that as carrier fluid, e.g.,oil, is withdrawn from the chamber 21 through the second outlet 20, asmaller trailing sample droplet 23 collides with the leading sampledroplet 22 so that the mixing occurs. FIG. 3 panel D shows a largermixed sample droplet 24 leaving the chamber 21 via the first outlet 19.

In more detail, initially, the entire system is primed with a densitymatched carrier fluid, e.g., oil. The diameter of the inlet 18 and theoutlets 19 and 20 is 250 μm. The spacing between the inlet 18 and theoutlet 19 is about 1 mm. The spacing between the inlet and outlet canrange from 0.2 mm to 1.5 mm. The carrier fluid is controlled to have apressure of about 0.5 bar to about 1.0 bar above atmospheric. Thecarrier fluid, e.g., silicone oil, has a viscosity of 0.08 to 0.1 Pas.

As with liquid bridges 1 and 9, sample droplets are enveloped by carrierfluid entering and exiting the bridge 17 via a protective film of thecarrier fluid firm around the sample droplets. This provides anon-contacting solid surface that prevents carryover contamination fromone sample droplet to the next sample droplet. The carrier fluid is usedas the control fluid and is density-matched with the sample plugs suchthat a neutrally buoyant environment is created within the chamber. Whentwo unmixed sample droplets arrive at the chamber in series from theinlet 18, the first droplet assumes a stable capillary-suspendedspherical form upon entering the chamber (FIG. 3, panel A). Thespherical shape grows until large enough to span the gap between theports, forming an axisymmetric liquid bridge (FIG. 3, panel B). Thesecond outlet 20 removes a flow of carrier fluid, e.g., oil, from thechamber causing the first sample droplet to slow and remain as aspherical shape at the first outlet 19. This allows time for a secondsample droplet to form a stable capillary-suspended spherical shape onentering the chamber 21. With the first sample droplet formed as aspherical shape at the outlet 19, and the second droplet formed as aspherical shape at the inlet 18, the first and second sample dropletscan form as one and create an axisymmetric liquid bridge (FIG. 3, panelC). The mixed droplet then exits through the outlet port 19 (FIG. 3,panel D).

In certain embodiments, the flow conditions should be adjusted such thatflow through the inlet 18 is greater than the flow through the secondoutlet 20. A typical flow through the inlet port 18 is about 5 μl/min,and can generally range from about 2 μl/min to about 7 μl/min. The flowaway from the chamber 21 through the second outlet 20 is typically 2.5μl/min and can generally range from about 1 μl/min to about 5 μl/min.Since there is conservation of mass flow within the bridge, this meansthat the flow through the first outlet 19 will balance the bridge togive a flow of typically 2.5 μl/min, and can generally range from about1 μl/min to about 5 μl/min.

In certain embodiments, the liquid bridge 17 can be used with a constantoutlet flow rate through the second outlet 20. In this embodiment,droplets can be mixed and the fluid flow through the system can bedecreased. In addition, liquid bridge 17 can be used in conjunction witha sensor to time the withdrawal of fluid through the second outlet 20 soas to maintain a generally constant sample flow rate.

The sensor used can be a droplet detection sensor that includes a LEDand photodiode. The LED is projected directly onto the center of thetube. A photodiode is positioned directly opposite the LED to pick upthe light refracted through the tube. As a sample droplet having varyingproperties compared to that of the carrier fluid, e.g., oil, flows pastthe LED and photodiode, the light refracted through the liquid isaltered slightly. This slight alteration is detected by the photodiodein the form of a change in voltage. This change in voltage can be usedto time the control flow through second outlet port 20.

Liquid bridge systems of the invention can further include at least onerobotics system to control the gas-free sampling devices. The roboticssystems control movement of the sampling device between wells of thefirst and second arrays and also control sample acquisition. At leastone pump is connected to the sampling device. An exemplary pump is shownin Davies et al. (WO 2007/091229, the contents of which are incorporatedby reference herein in their entirety). Other commercially availablepumps can also be used. The pumps are controlled by a flow controller,e.g., a PC running WinPumpControl software (Open Cage Software, Inc.,Huntington, N.Y.), for control of direction of flow and flow rates.

Liquid bridge systems can be fluidly connected, e.g., tubes or channels,to an type of analysis device. In certain embodiments, the liquid bridgesystem is connected to a thermocycler to perform PCR reactions on theacquired sample. An exemplary thermocycler and methods of fluidlyconnecting a thermocycler to a liquid bridge system are shown in Davieset al. (WO 2005/023427, WO 2007/091230, and WO 2008/038259, the contentsof each of which is incorporated by reference herein in its entirety).The thermocycler can be connected to an optical detecting device todetect the products of the PCR reaction. An optical detecting device andmethods for connecting the device to the thermocycler are shown inDavies et al. (WO 2007/091230 and WO 2008/038259, the contents of eachof which is incorporated by reference herein in its entirety).

The invention having now been described, it is further illustrated bythe following examples and claims, which are illustrative and are notmeant to be further limiting. Those skilled in the art will recognize orbe able to ascertain using no more than routine experimentation,numerous equivalents to the specific procedures described herein. Suchequivalents are within the scope of the present invention and claims.

The contents of all references and citations, including issued patents,published patent applications, and journal articles cited throughoutthis application, are hereby incorporated by reference in theirentireties for all purposes.

EXAMPLES Example 1 Rupturing of a Sample in a Liquid Bridge

Liquid bridge stability was studied as a means to predicting thegeometric conditions at which rupture occurs. Liquid bridge rupture maybe defined as the complete breakage of the liquid filament connectingone solid support to the other. The dimensionless parameterscharacterizing liquid bridges are used to define the stability boundaryat which rupture was observed. FIG. 7 presents images of liquid bridgesat three slenderness conditions just prior to rupture. The rupture wascaused by the withdrawal of liquid bridge fluid from one capillary tube.It was observed that low slenderness ratio liquid bridges, an example ofwhich is shown in FIG. 7, panel A, adopt a thimble shape at the minimumvolume stability. Larger slenderness ratio liquid bridges, such as thatshown in FIG. 7, panel C, possess a barrel form with a maximum radius atthe bridge mid-span. Intermediate slenderness ratios were found to havea near cylindrical shape at the minimum volume stability limit. FIG. 7,panels A-C show liquid bridges with slenderness ratios of 1.09, 2.45 and6.16 respectively.

Example 2 Stability of a Liquid Bridge with Respect to Slenderness andVolume

The stability of liquid bridges was examined as a function ofslenderness, Λ*, which is the ratio of tip separation, L, to the meandiameter, 2R₀, of the supporting capillaries, i.e. A*=L/2R₀. Stabilitywas also investigated as a function of volumetric ratio, V*, which isthe ratio of liquid bridge volume to the volume of a cylinder with aradius R₀, the average radius of the supporting capillaries, i.e.:

V*= V /(πR ₀ ² L).

The location of the stability boundary, or rupture point, was determinedexperimentally by fixing the slenderness, establishing a stable liquidbridge between capillary tips and withdrawing fluid from one capillaryuntil rupture was observed. A digital image of the liquid bridge justprior to rupture was then analyzed, using an edge detection measurementtechnique to determine the total volume and hence the volumetric ratio,V*. The slenderness was then adjusted and the experiment repeated. K*represents the ratio of the radius of the smaller disk, R₁, to theradius of the larger one, R₂, that is K*=R₁/R₂. FIG. 10 shows theapproximate location of the minimum volume stability boundary for liquidbridges with a lateral Bond number of 1.25×10⁻⁴, a near weightlessenvironment. Vertical and horizontal error bars indicate experimentaluncertainty.

At high volumetric ratios, FIG. 7 panel C for example, bridges maintaintheir integrity and reach a minimum energy configuration. At lowvolumetric ratios, FIG. 7 panel A for example, the bridges break beforethe interfacial energy is minimized. The initial dip in the stabilityboundary at low slenderness ratios was caused by low-volume droplets notfully wetting the exposed fused silica of the capillary tips. Theinfluence of unequal capillaries on the Λ*−V* stability diagram is alsoshown in FIG. 8. It can be seen that the unstable region of the Λ*−V*plane increases as the parameter K*, the ratio of capillary radii,decreases. The results presented in FIG. 8 confirmed that the staticstability of liquid bridge is purely geometrical at low Bond numbers. Itis notable that low slenderness ratio bridges are almost completelystable, with respect to rupture, for all capillary radii measured.

Rupture was observed only at very low volumetric ratios with the liquidbridge assuming a thimble shape. Liquid bridge instability when appliedto fluid dispensing is particularly useful as a replacement formicro-channel shear-based dispensing systems. In more detail, FIG. 8shows a stability diagram for a de-ionized water liquid bridge in adensity matched silicone oil, Bond number: 1.25×10⁻⁴. Vertical errorbars indicate the volumetric ratio uncertainty as a result of cameraframe rate. Horizontal error bars indicate slenderness uncertainty dueto capillary tip misalignment. The parameter K* is the ratio ofsupporting capillary radii.

Example 3 Dispensing Sub-Microliter Volumes

The following describes the use of liquid bridge instability as amechanism for dispensing sub-microliter volumes of fluid in a continuousmanner. The dispensing mechanism provided a reliable means of producinguniform aqueous plugs separated by silicone oil that did not rely on theshear force exerted by the carrier fluid. The repeatability with whichthe method dispensed plugs was examined. The approach used the liquidbridge's dependence on geometry to create a periodic instability betweenopposing capillary tips. A stable liquid bridge was first establishedbetween aqueous inlet and outlet. The volume held in this bridge wasthen steadily reduced by the action of the silicone oil inlet. Thiscaused the formation of an unstable liquid bridge that ruptured torelease a smaller plug at the outlet. The segmenting mechanism provideda reliable means of producing uniform aqueous plugs separated bysilicone oil that did not rely on the shear force exerted by the carrierfluid. Furthermore, a protective oil film was established between thewalls of the circular capillaries and the droplet to prevent carryovercontamination.

FIG. 9 panels A-D shows images of a liquid bridge dispensing at fourdifferent slenderness ratios. (A) Λ*=0, (B) Λ*=0.76, (C) Λ*=1.37 and (D)Λ*=2.31. Q*=0.5, K*=0.44. Increasing the capillary tip separation, andhence the slenderness ratio increased the plug volumes dispensed. Q*,the oil flow rate as a fraction of the total flow rate, was maintainedconstant at 0.5. FIG. 9 panel A shows dispensing with the dispensingcapillary inserted inside the outlet capillary. This configuration wasassigned a slenderness ratio, Λ* of zero. Slenderness ratios close tozero resulted in the smallest volume plugs dispensed for this geometry.The effect of increasing tip separation on dispensed plug volume isshown in FIG. 9 panels B-D. Increasing tip separation, i.e. slendernessratio, resulted in larger volume aqueous plugs punctuated byapproximately the same volume of silicone oil. This was due to thesilicone oil inlet flow rate being maintained constant and equal to theaqueous droplet inlet flow rate.

FIG. 10 presents a plot of V*, against slenderness ratio, Λ*, where V*is the dimensionless plug volume scaled with R₀ ³, i.e.:

V*= V/R ₀ ³.

Results are presented for three different values of the oil flow ratefraction, Q*, with the ratio of capillary tip radii, K*, maintainedconstant at 0.44. The axis on the right-hand side of the plot indicatesthe measured plug volume. Horizontal error bars indicate slendernessuncertainty as a result of positional inaccuracy. Vertical error bar area result of uncertainty in the plug volume calculation due to imageprocessing. The results show the expected trend of increased plug volumewith liquid bridge slenderness ratio. Decreasing Q* resulted in adramatic increase in dimensionless plug volume. Altering Q* alsoaffected the volume of silicone oil separating the aqueous plugs as Q*is the oil flow rate as a fraction of the total flow rate. The lowestrepeatable volume measured using this particular geometry wasapproximately 90 mL with Λ*=0, Q*=0.75. The highest volume measured wasapproximately 3.9 μL with Λ*=2.36, Q*=0.25.

In flows where the non-wetting fluid, i.e. the aqueous phase, wasdisplaced by wetting fluid, i.e. oil, a thin film of the wetting fluidseparated the droplets from the capillary surface. The thickness of thefilm resulted from a balance between the oil viscosity, η, and theinterfacial tension, σ_(i). The thickness of the oil film deposited in acapillary of radius r is given by;

h=1.34 r(Ca ^(2/3)).  (Equation (0.1)

The capillary number, Ca, is given by:

Ca=ΘU/σ _(i),  (Equation (0.2)

where U represents the mean velocity of the flow. Equation (0.1) isobeyed if the film is thin enough to neglect geometric forces, h<0.1 r,and thick enough to avoid the influence of long range molecularattraction, h>100 nm. Typical oil film thicknesses for plug flow through400 μm polymeric fluorocarbon internal diameter tubing were calculatedto be of the order of 1 μm.

This film thickness was too small to resolve with any degree of accuracyfrom experimental images. However, the oil film did form a protectivecoating preventing aqueous reactor fluid from contacting the Teflontubing. This had the advantage of preventing a mechanism responsible forcarryover contamination whereby small droplets may be deposited onto thewalls of micro-channels. Table 1 below presents two examples ofoil-surfactant combinations that were used to successfully establishprotective oil films around flowing droplets. Surfactant additives actedto change the interfacial tension between droplets and the oil carrierfluid such as to promote the establishment of a protective oil film, thethickness of which is given by Equation 0.1.

TABLE 1 Oil Surfactant Concentration FC40 1H,1H,2H,2H-perfluoro- 2% W/V1-decanol AS100 Triton X-100 0.1% W/W in PCR Silicone Oil BufferSolution

FIG. 11 presents a dimensionless plot of the product of V* and Q* versusΛ*. The data, taken from the plot shown in FIG. 10, collapsed on to thetrend line within the bounds of uncertainty. The data applied togeometries with K*=0.44. Notwithstanding this geometric constraint, thecollapsed data did yield valuable design information.

Consider a microfiuidic system designer deciding on an appropriategeometry for a segmenting device. The designer will usually know theexact volume to dispense from the outline specification for the device.If there is a sample frequency requirement, the designer may also know avalue for Q*. Recalling that K*=R₁/R₂, where R₁ and R₂ are the inlet andoutlet diameters respectively makes the design process relatively easy.Deciding on an arbitrary value for an outlet diameter fixes the aqueousinlet diameter as the data shown in FIG. 12 applies to only togeometries with K*=0.44. With this information in hand, an appropriatevalue for V*(Q*) may be calculated. The corresponding value for Λ* maythen be read from the design curve shown in FIG. 11. Finally, Λ* wasused to calculate the tip separation between the inlet and outlet.

Example 4 Droplet Volume with Respect to Liquid Bridges

The data presented in FIGS. 10 and 11 applied to geometries withK*=0.44. The effect of altering K* on plug volumes dispensed was alsoinvestigated. FIG. 13 panels A-C shows a liquid bridge dispensing atthree different values for K*. Panels (A), (B) and (C) correspond to K*values of 0.25, 0.44 and 1.0 respectively. K* value of 0.25 was achievedby assembling a 200 μm fused silica microcapillary at the end of apolymeric capillary tube by a reduction of internal diameter throughappropriately sized fused silica. Sealing was ensured with the additionof cyanoacrylate glue at the sleeve interfaces.

FIG. 12 presents a dimensionless plot of V* versus Λ* for threedifferent values of K*. The dimensionless plug volume, V*, was scaledwith R₂ ³, and not R₀ ³ as previously. This permitted a directcomparison of dimensionless plug volumes as R₂ remained constantthroughout the experiment. It was observed that decreasing K* generallylowered the plug volumes dispensed for any given value of slenderness,Λ*. The minimum volume dispensed with K*=0.25 was approximately 60 mLwhilst that of K*=0.44 and K*=1 was approximately 110 mL. Attempts tocollapse the data shown in FIG. 12 onto a single line, similar to theplot shown in FIG. 1, were unsuccessful. This was due to the highlynon-linear relationship between K* and V* for any given value of Λ*.

Example 5 Repeatability of Dispensing Sub-Microliter Volumes

The repeatability with which the liquid bridge dispensing system coulddeliver fluid was of particular interest. FIG. 14 plots plug volumevariation over fourteen measurements for a dispensing system withK*=0.44. The results show mean plug volumes of approximately 120 mL and56 mL with maximum volumetric variations of ±4.46% and ±3.53%respectively. These volumetric variations compared favorably tocommercial available micropipettes that have an uncertainty of ±12% whendispensing 200 mL. The accuracy with which one may dispense usingmicropipettes, however, is thought to be largely dependant upon userskill. The automation of dispensing systems may therefore be justifiedas a means of eliminating user-user variability. The volumetric analysispresented in FIG. 14 shows liquid bridge dispensing to be a veryrepeatable means of continuously dispensing sub-microliter volumes offluid.

FIG. 15 is an image of a liquid bridge. The bridge consisted of twoopposing capillaries of the same external diameter. The second inletpart was of a finer capillary orientated at right angles to and situatedhalf-way between the other two capillaries. Constraints on opposingcapillary radius and the placement of the third capillary helped tosimplify the dimensionless stability study. The investigation alsonecessitated modifications to the dimensionless parameterscharacterizing axisymmetric liquid bridge geometry. The slendernessratio, A*, was calculated using:

$\begin{matrix}{{\Lambda^{*} = \frac{\sqrt{L^{2} + S^{2}}}{2R_{0}}},} & {{Equation}\mspace{14mu} (1)}\end{matrix}$

where L and S correspond to the distances indicated in FIG. 15. R₀ isdefined as the mean radius, i.e. (R₁+R₂)/2. K* is defined as R₁/R₂. Thevolumetric ratio, V*, is defined as:

$\begin{matrix}{{V^{*} = \frac{\overset{\_}{V}}{\pi \; R_{0}^{2}\sqrt{L^{2} + S^{2}}}},} & {{Equation}\mspace{14mu} (2)}\end{matrix}$

where V is the measured volume at which bridge collapse occurs. In termsof the geometry presented in FIG. 15, a funicular bridge collapsecorresponded to detachment from the finer capillary.

FIG. 16 shows a stability diagram for the approximate location of theminimum volume stability boundary for purified water funicular liquidbridges with a lateral Bond number of 1.25×10⁻⁴, a near weightlessenvironment. The boundaries of stability were found by fixing a valuefor Λ*, establishing a stable funicular bridge and withdrawing fluiduntil the bridge collapsed. The collapse was recorded via a CCD and theframe immediately following rupture was analyzed to measure the volume.The calculation of the bridge volume was simplified by the fact that thecollapsed funicular bridge exhibited axisymmetry with respect to theaxis of the two larger capillaries. Minimum volume stability boundarieswere plotted for K*=0.25 and K*=0.44. Lower K* values displayedincreased instability. Volumetric data for Λ* values lower thanapproximately 1.5 were difficult to obtain with the geometry used and sowere omitted from the stability diagram.

The formation of a funicular bridge deemed unstable by the graph shownin FIG. 16 ensured the injection of fluid into an aqueous plug passingthrough opposing capillaries. A further advantage to using funicularbridge dispensers is based on the speed at which the process takesplace. Typical instabilities last of the order of 100 ms, insufficienttime for the host droplet fluid to diffuse to the dispensing capillarytip. This is a further preventative measure against carryovercontamination.

The two input one output, funicular bridge can be configured so that theexpression profile of many genes may be addressed. One input containsthe primer and premix in a continuous phase, the outlet then deliversthem in droplet form. Firstly many input and output capillaries, say p,can be set in planes perpendicular to that of FIG. 1. A perpendiculararrangement allows for good optical access in the planar thermocyclerwhich is connected to the output. Each arrangement of two inputs and oneoutput can be used to address a single primer, giving p primers. This,however, would make for a very long device in the plane perpendicular toFIG. 1. If serially variant primers were fed into each input, numberingq, this would reduce the scale. Further, if the primers weremultiplexed, to order r, in each droplet the scale would be furtherreduced. The number of primers that could then be addressed would be:N=p×q×r. By this means, a PCR test of the whole genome of any livingform, including the human, could be addressed, which would haveapplications beyond diagnosis, in many fields of pure and appliedscience.

1. A method of constructing a liquid bridge system comprising assemblinga liquid bridge system comprising a predetermined number of liquidbridges sufficient to combine a first sample array and a second samplearray for analysis.
 2. The method according to claim 1, furthercomprising ascertaining a number of wells within the first array ofsamples, and ascertaining a number of wells within the second array ofsamples.
 3. The method according to claim 2, further comprising applyinga formula a_(n)×b_(n), wherein a_(n) is the number of wells in the firstarray and b_(n) is the number of wells in the second array.
 4. Themethod according to claim 1, wherein the first and second array ofsamples are each independently chemical or biological species.
 5. Themethod according to claim 1, wherein the first array of samples isprimers for PCR reactions and the second array of samples comprisesnucleic acid.
 6. The method according to claim 1, wherein the nucleicacid is DNA or cDNA.
 7. A liquid bridge system constructed by theprocess of claim
 1. 8. A method for forming predetermined samplecombinations comprising: providing a first sample array; providing asecond sample array; and providing at least one liquid bridge to mix thefirst sample array with the second sample array, wherein the number ofliquid bridges provided is determined by a formula a_(n)×b_(n) whereina_(n) is the number of wells in the first array and b_(n) is the numberof wells in the second array.
 9. The method according to claim 8,wherein the first and second array of samples are each independentlychemical or biological species.
 10. The method according to claim 8,wherein the first array of samples is primers for PCR reactions and thesecond array of samples comprises nucleic acid.
 11. The method accordingto claim 10, wherein the nucleic acid is DNA or cDNA.
 12. The methodaccording to claim 8, wherein each of the first array and the secondarray is independently a 96 well plate or 384 well plate.
 13. The methodaccording to claim 8, wherein at least one of the predetermined samplecombinations is a combination of a blank from the first array and ablank from the second array.
 14. The method according to claim 13,wherein the blank in each of the first and second array is oil.
 15. Asystem for mixing samples, the system comprising, a first gas-freesampling device that interacts with a first sample array; a secondgas-free sampling device that interacts with a second sample array; andat least one liquid bridge for mixing the first sample array with thesecond sample array, wherein the number of liquid bridges provided isdetermined by a formula a_(n)×b_(n), wherein a_(n) is the number ofwells in the first array and b_(n) is the number of wells in the secondarray.
 16. The system according to claim 15, further comprising roboticsto move the first and second sampling devices to interact with the firstand second arrays of samples.
 17. The system according to claim 15,further comprising pumps for acquiring samples in the first and secondarrays.
 18. The system according to claim 15, further comprising acomputer operably connected to the system.
 19. The system according toclaim 15, further comprising a thermocycler.